Borrelia burgdorferi, the causative agent of Lyme disease, establishes persistent infection that can affect the joints, heart, skin, and nervous system. The ability of this spirochete to establish infection demands 1) dissemination from the site of inoculation by an infected tick, 2) adaptation to the mammalian environment, and 3) persistence despite the host immune response. Interactions with mammalian cells occur continually during each of these steps and phases of infection. Using a phage display selection in vitro, we showed that the B. burgdorferi outer membrane protein, P66, binds to the 3-chain integrins. In an in vivo phage display selection, we also showed that OspC serves as an adhesin to an unknown receptor in the endothelium in living mice. Phage clones bearing OspC sequences were the single group most highly selected in vivo, and most of the clones were selected in the heart. OspC is a protein of the Lyme disease Borrelia that is expressed as the bacteria move from tick to mammal, and was previously shown by another group to be critical for the earliest stages of infection of mice. A different group showed that a deficiency in OspC could be partially overcome in scid mice by over-expression of other lipoproteins. This work also revealed that OspC does appear to be essential for normal dissemination of the bacteria to the heart, the tissue in which our phage clones were most frequently selected. Our data are also consistent with previous work by a third group demonstrating that B. burgdorferi in the heart express higher levels of ospC than do the bacteria in other tissues tested. Our overall hypothesis is that the adhesin activity of OspC is important to its critical function in the life of B. burgdorferi. Based on our results as well as those of the three other groups, we will test two facets of this hypothesis in this proposal. The first is that OspC recognizes a receptor localized in regions of the heart colonized by B. burgdorferi. The second is that specific conserved amino acids are required for adhesin activity and normal function in establishment of disseminated infection in mice. B. burgdorferi strains that do and do not express OspC will be tested for adhesion activity in cell culture and targeting of the heart, and particular regions therein, in mice. Additional strains expressing mutated alleles of OspC will be generated and tested using the same approaches. In this set of experiments we propose to employ our unique tools to further define, at the biochemical level, the role of OspC in B. burgdorferi infection. We anticipate that this work may enable the design of therapeutic approaches to halt dissemination of B. burgdorferi after the tick bite even in unvaccinated people and animals. PUBLIC HEALTH RELEVANCE: Lyme disease is now the most prevalent vector-borne illness in the northern hemisphere, and a significant burden on the health system in regions in which it is common due to neurologic, cardiac, and rheumatologic manifestations of disease. We propose to delve into how Borrelia burgdorferi interacts with the heart in a mammalian host. We will focus on one B. burgdorferi protein, OspC, which is essential for the bacteria to cause the normal disseminated infection in mice. Our overall goal is to understand how OspC recognition of host cell molecules allows B. burgdorferi to overcome host barriers to the establishment of persistent, disseminated infection, which could eventually lead to strategies that will help the host immune system clear the organism as adjuncts to antibiotic therapy.